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Conclusions/Significance

These results demonstrate for the first time that the IL-10-producing B10 B cell subset has the capacity to suppress IgG humoral immune responses against both foreign and self antigens. Thereby, therapeutic agents that drive regulatory B10 cell expansion in vivo may inhibit pathogenic IgG autoAb production in humans.

Introduction

Interactions between CD40 expressed by B cells and its ligand CD154, expressed by antigen (Ag)-activated CD4+ helper T cells, promotes BCR-induced B cell proliferation and survival, which is essential for isotype switching and germinal center (GC) formation [1], [2], [3], [4], [5], [6]. Interrupting CD40 and CD154 interactions prevents the development of both T cell-dependent (TD) humoral immune responses and cell-mediated immune responses [7]. Agonistic CD40 mAbs are also potent immune adjuvants for both short-lived humoral-immunity to T cell-independent Ags [8], [9] and cellular immune responses to viruses and tumors [10], [11], [12]. However, CD40 agonists given during TD immune responses actually ablate GC formation, induce a pattern of extrafollicular B cell differentiation in the spleen and lymph nodes, prematurely terminate humoral immune responses, block the generation of B cell memory, and prevent the generation of long-lived bone marrow plasma cells [13]. Consistent with this, ectopic CD154 expression by B cells in transgenic mice (CD154TG) terminates germinal center responses prematurely and leads to augmented plasma cell production in T cell areas [14], [15]. Expression of the CD154 transgene in these mice is driven by immunoglobulin (Ig) gene promoter and enhancer elements, resulting in B cell-specific expression [14], [15]. B cell CD154 expression has a precedent in human disease, as it is expressed by both T cells and B cells in systemic lupus erythematosus (SLE) patients and in a mouse model of lupus [16], [17], [18], with ectopic B cell expression of CD154 in aged hemizygous CD154TG mice leading to intestinal inflammation [19] or SLE-like autoimmunity including anti-DNA autoAbs and glomerulonephritis [20]. While a certain level of B cell CD40 signaling can exacerbate the development or severity of autoimmune disease, these studies collectively suggest that the fate of Ag-specific B cells is dramatically altered by the extent of CD40 ligation, with heightened CD40 signaling potentially representing a physiological means to limit the duration and intensity of immune responses.

CD22 negatively regulates transmembrane signals in B cells through association with the potent intracellular phosphatases SHP-1 and SHIP [21], [22], [23], [24]. B cells from CD22−/− mice are markedly hyper-responsive to CD40 signals, whereby their ex vivo stimulation with agonistic CD40 mAb induces a much greater degree of proliferation relative to wild type (WT) B cells [25]. As such, potent in vivo signals provided by constitutive CD40 signaling combined with CD22 deficiency may alter the duration and intensity of immune responses, size of the autoreactive B cell pool, and autoAb production levels. To test this, CD22−/− mice homozygous for the CD154 transgene (CD154TGCD22−/−) were generated. Remarkably, the defining in vivo characteristic of CD154TGCD22−/− mice was a dramatic expansion in regulatory B10 cells that were competent to express IL-10 [26], [27], and meager IgG production against both foreign and self Ags. Thus, enhancing CD40 signaling limited the duration and intensity of humoral immune responses likely by driving the expansion of B10 cells, a B cell subset that is also found in humans [28]. Inducing such an expansion of B10 cells may be particularly therapeutic in autoimmune syndromes such as SLE where aberrant CD154 expression contributes to inflammation and the generation of pathogenic isotype-switched B cells.

Methods

Ethics statement

All animal studies and procedures were approved by the Duke University Institutional Animal Care and Use Committee (approved IACUC protocol #A008-08-01; Duke University PHS Animal Welfare Assurance No. A3195-01).

Mice

CD22−/− mice, backcrossed with C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) for ≥8 generations were previously described [25]. CD154 transgenic mice [20] were crossed to homozygosity and referred to as CD154TG mice. Double mutant mice were generated by interbreeding the F1 offspring of CD22−/− and CD154TG mice, with CD154TGCD22−/− mice maintained as homozygous at both genetic loci by sibling matings. C57BL/6 WT control mice were purchased from either The Jackson Laboratory (Bar Harbor, ME) or NCI Frederick (Bethesda, MD). Bcl-xL transgenic mice [29] were a kind of Dr. Michael Farrar (University of Minnesota, Minneapolis, MN). Unless otherwise indicated, all mice used in these studies were between 8 and 14 weeks of age. Mice were housed in a specific pathogen-free barrier facility.

Experimental autoimmune encephalomyelitis (EAE) experiments

Purified spleen B cells from CD154TGCD22−/− donor mice were surface labeled with CD1d and CD5 mAbs, with CD1dhiCD5+ and CD1dloCD5− B cell populations isolated using a FACSVantage SE flow cytometer with purities of 95–98%. 1×106 cells were either directly transferred i.v. into WT recipient mice, or were first cultured with agonistic CD40 mAb for 48 h with LPS added during the final 5 h of culture, then washed and transferred. One day after cell transfers, EAE was induced in recipient mice with clinical signs of EAE scored daily as described [30], [31].

Immunizations

In some experiments, WT, CD22−/−, CD154TG, and CD154TGCD22−/− mice were immunized i.p. with 100 µg DNP-KLH in CFA, and then boosted on day 21 with DNP-KLH in IFA. Serum Ag-specific Ig levels were determined by ELISA. Alternatively, the mice were immunized i.p. with 50 µg 4-hydroxy-3-nitrophenyl acetyl-conjugated chicken γ-globulin (NP8CGG) in alum, with spleens were harvested on day 10 and the splenocytes stained with GL-7 and B220 mAbs, with double-positive B cells identified by flow cytometry. In other experiments where adjuvant was excluded, WT mice were immunized i.p. with 100 µg DNP-KLH in PBS alone, and then boosted on day 21 with DNP-KLH in PBS alone.

The CD1dhiB220+ marginal zone (MZ) B cell population was significantly reduced in CD22−/− mice as assessed in frozen spleen sections (Figure 1D), as published [24], [34], [35]. Remarkably, the MZ B cell population was restored in CD154TGCD22−/− mice, revealing that CD40 ligation overcomes the effects of CD22 deficiency on MZ maintenance. Nevertheless, CD1dhiB220+ cells were also observed within B cell follicles of all genotypes. CD1dhi B cells were particularly abundant within the MZ and follicles of CD154TGCD22−/− mice (highlighted in the enlarged region), paralleling their overall high numbers relative to the other mouse lines (Figure 1E).

CD22−/− B cells proliferate much more extensively than WT B cells when cultured with agonistic CD40 mAb [25]. B cells from CD154TG mice proliferated less than WT B cells following CD40 stimulation as quantified by [3H]-thymidine incorporation (Figure 1F), suggesting desensitization of the CD40 pathway in these mice. However, CD154TGCD22−/− B cells proliferated more extensively than WT B cells following CD40 stimulation, indicating that the hyper-responsiveness caused by CD22 deficiency persists in these cells. Altered CD40 expression does not explain these results, as cell surface CD40 expression was equivalent on spleen B cells from WT, CD22−/−, CD154TG and CD154TGCD22−/− mice as determined by immunofluorescence staining with flow cytometry analysis (data not shown). Similar to CD40 ligation, proliferation of CD154TGCD22−/− and CD22−/− B cells in response to LPS stimulation was modestly enhanced relative to WT or CD154TG B cells as quantified by CFSE dilution analysis (Figure 1G).

In contrast to CD40 stimulation, CD22−/− B cells undergo apoptosis following extensive BCR ligation due to blocked cell cycle progression [25]. Likewise, proliferation of B cells from CD154TGCD22−/− mice was profoundly deficient following BCR ligation with F(ab)′2 anti-IgM Ab and nearly identical to the defect of CD22−/− B cells (Figure 1F). Without CD40 or BCR stimulation, background levels of B cell [3H]-thymidine incorporation were low, but were significantly higher (P<0.01) for B cells from CD154TGCD22−/− and CD154TG mice than for WT and CD22−/− mice (data not shown). Consistent with this, purified B cells from both CD154TG and CD154TGCD22−/− mice remained viable when cultured for >10 days without any exogenous mitogenic stimulation, while CD154TGCD22−/− B cells uniquely proliferated at significant levels as assessed by CFSE dilution (Figure 1H). Collectively, these data demonstrate that B cells in CD154TGCD22−/− mice are capable of receiving CD40 and additional costimulatory signals that promote B cell activation, survival, and proliferation.

Importantly, transgenic CD154 engagement of CD40 in CD154TG and CD154TGCD22−/− mice appears to be primarily mediated through cis (same cell) interactions on B cells, since mixed bone marrow chimeras derived from WT and CD154TG donors exhibit enhanced Ab production specifically from CD154TG B cells [14], [15]. Also, activation markers on dendritic cells are not altered in CD154TG mice relative to WT mice [14], [15]. In addition, despite being hyper-responsive to stimulation with agonistic CD40 mAb, CFSE-labeled CD22−/− spleen B cells co-cultured with CD154TGCD22−/− spleen B cells ex vivo did not exhibit enhanced survival or proliferation compared to CD22−/− B cells cultured alone (data not shown). These observations strongly suggest that the phenotypic alterations observed in CD154TG and CD154TGCD22−/− mice are mediated by enhanced, autocrine CD40 signaling in the B cell lineage, and not through the activation of bystander cells in other lineages that also express CD40.

Reduced IgG Ab and autoAb production in CD154TGCD22−/− mice

Serum IgM concentrations were significantly higher in both CD22−/− (155% increase) and CD154TG (125% increase) mice at 4 mos of age when compared with WT mice, but were dramatically higher (1,220% increase) in CD154TGCD22−/− mice (Figure 2A). At 12 mos of age, IgM levels were equally high in both CD154TGCD22−/− and CD22−/− mice. By contrast, serum IgG levels were reduced in both CD154TGCD22−/− and CD22−/− mice at 4 mos of age, and remained significantly reduced in 12 mo-old CD154TGCD22−/− mice relative to all other genotypes. Thus, IgM levels were high in CD154TGCD22−/− mice during early life, with little IgG Ab produced throughout life.

IgM autoAbs reactive with dsDNA, ssDNA and histone protein were significantly higher in both CD154TGCD22−/− and CD22−/− mice at 12 mos of age, while CD154TG mice only had significant levels of IgM autoAbs reactive with dsDNA (Figure 2B). CD22−/− mice also generated significant levels of IgG autoAbs reactive with dsDNA and ssDNA, but IgG autoAbs were virtually absent in CD154TGCD22−/− mice. Therefore, CD154TGCD22−/− mice produced IgM but not IgG autoAbs.

Following immunization with the TD Ag DNP-KLH in Freund's adjuvant, WT, CD22−/−, and CD154TG mice generated significantly stronger DNP-specific IgG responses than CD154TGCD22−/− mice (Figure 2C). Remarkably, classical GC structures were absent in the spleens of both CD154TG and CD154TGCD22−/− mice after the boost phase of DNP-KLH immunization as assessed by in situ immunofluorescence staining of frozen spleen sections (Figure 2C). Nevertheless, relatively rare GL7+B220+ B cells were detectable within the B cell follicles of spleens from CD154TG mice, but not within the spleens of CD154TGCD22−/− mice (Figure 2C, enlarged regions). Despite lacking classical GCs, the presence of detectable GL7+B220+ B cells in CD154TG spleens (Figure 2C–D) coupled with their 4-fold spleen B cell expansion (Table 1) suggests that enough “GC-like” B cells are present to produce significant isotype switched IgG antibody. Similar results were obtained from mice immunized using the TD Ag NP8-CGG in alum, followed by quantitative analysis of total spleen GL7+B220+ B cells by flow cytometry. The frequency of spleen GL7+ B cells was significantly reduced in both CD154TGCD22−/− (90% reduced) and CD154TG (77% reduced) mice relative to WT mice after immunization (Figure 2D, contour plots). However, the total number of GL7+ B cells induced following immunization was specifically reduced in CD154TGCD22−/− mice (62%, bar graphs), remaining normal in CD154TG mice as a result of their expanded B cell pool. Thus, global IgG immune responses were specifically impaired in CD154TGCD22−/− mice.

A fraction of spleen CD1dhiCD5+ B cells, called B10 progenitor (B10pro) cells, is induced to become IL-10 competent during 48 h cultures with agonistic CD40 mAb [27]. Since it is not yet possible to phenotypically distinguish existing B10 cells from in vitro matured B10pro cells, IL-10-producing B cells under these conditions are collectively defined as ‘B10+B10pro’ cells. Spleen B10+B10pro cell numbers in CD154TGCD22−/− mice were 11-, 12-, and 2.8-fold higher than in WT, CD22−/−, and CD154TG mice, respectively (Figure 3C and Table 1).

B10 cells in CD154TGCD22−/− mice are regulatory

Spleen B10 cells localize primarily within the CD1dhiCD5+ subpopulation in WT mice (Figure 4A) as described [37]. Likewise, spleen B10 cells in CD154TGCD22−/− mice were found primarily within the CD1dhiCD5+ population (Figure 4A), where they represented >50% of the cells (bar graphs). This was >3 times the 15% frequency of B10 cells found within the CD1dhiCD5+ population of WT mice, and far above the 4% frequency of B10 cells found within the CD1dloCD5− subpopulation of CD154TGCD22−/− mice.

Discussion

CD154 is expressed at relatively high levels by both T cells and B cells in SLE patients and in a mouse model of lupus, which is proposed to drive CD40 signaling, B cell hyperactivity, and autoAb production [16], [17], [18]. Although CD154 expression by B cells in the current study appears lower by comparison, the combination of CD154 expression and CD22 deficiency significantly enhanced B cell responses to CD40 signaling (Figure 1). B cell CD154 expression in CD154TGCD22−/− mice also led to a remarkable 16-fold expansion of the regulatory B10 cell subset relative to WT mice. B10 cells normally represent only 1–3% of spleen B cells in WT mice [30], [37]. In fact, B10 cells alone represented 16% of spleen B cells in CD154TGCD22−/− mice, while B10+B10pro cells represented 39% of spleen B cells. B10 cell expansion also paralleled a dramatic reduction in B cell isotype switching, with lower IgG Ab and autoAb levels in CD154TGCD22−/− mice than were present in the parental mouse lines, even after immunization using a strong TD Ag in adjuvant. IgG deficiency was not observed in CD154TG mice where spleen B10 cells were only expanded 3.8-fold and total spleen B cells were expanded 3.6-fold (Figure 3A and Table 1). Thus, heightened CD40 signaling through the combination of chronic CD154 expression and CD22-deficiency drove the in vivo expansion of regulatory B10 cells with functional activity, and limited the intensity of IgG immune responses, both of which may limit the pathogenic consequences of autoimmunity.

B cell negative regulation of immune responses through the production of IL-10 has been demonstrated in EAE [30], [39], [40], [41] and other mouse models of autoimmunity and inflammation [30], [36], [42], [43], [44], [45]. These B cells are called “B10 cells” because IL-10 secretion is required for their negative regulatory function, and other B cell subsets with their own unique regulatory properties may also exist [26]. B10 cells have a potent capacity to down-modulate immune responses during adoptive transfer experiments [30], [37], [38], [46], a property clearly retained by CD154TGCD22−/− B10 cells (Figure 4C). Spleen B10 cells are predominantly contained within the phenotypically unique CD1dhiCD5+ subpopulation and are presumed to be functionally mature since they are competent to express quantifiable IL-10 after 5 h of ex vivo stimulation [30], [37]. B cell regulatory functions are also enhanced by ex vivo CD40 or LPS stimulation in inflammation and autoimmunity models [40], [47], [48], [49], although CD40 or TLR signaling are not required for B10pro or B10 cell generation [27]. However, CD40 signaling and LPS exposure can induce mouse B10pro cells to mature into functional IL-10-competent B10 cells [27], while LPS or CpG exposure can induce B10 cells to secrete IL-10. Thus, B10pro and B10 cells provide a fundamental linkage between the adaptive and innate immune systems. B10 cells in CD154TGCD22−/− mice were phenotypically and functionally similar to B10 cells in WT mice, including their characteristic high levels of CD19 expression and ability to respond to LPS and CD40 signals (Figure 3; [37]). The current studies clearly demonstrate that enhanced B cell CD40 signaling in vivo leads to a remarkable phenotypic outcome characterized by B10 and B10pro cell expansion, with suppressed isotype switching and IgG autoAb production that may be attributable to the regulatory activity of these cells through their production of IL-10.

Depleting the relatively small number of endogenous B10 cells in wild type mice significantly enhanced isotype switching and IgG production in response to immunization in the absence of adjuvant (Figure 5C–D). B10 cell depletion by CD22 mAb also enhances cellular immunity during EAE initiation [31]. By contrast, expanded B10 cell numbers in CD154TGCD22−/− mice paralleled their IgG-deficient phenotype. Further supporting a mechanistic explanation for reduced IgG production in CD154TGCD22−/− mice, B10 cells from CD154TGCD22−/− mice retained their regulatory function and suppressed EAE during adoptive transfer experiments (Figure 4C). It was not possible to directly compare the functional activities of B10 cells from WT and CD154TGCD22−/− mice in vivo because of their different relative frequencies within the CD1dhiCD5+ B cell subsets used for adoptive transfer experiments (Figure 4A), the fact that it is not possible to quantify Ag-specific B10 cell frequencies, and that B10 cells in CD154TGCD22−/− mice are not amenable to CD22 mAb-induced depletion. Nonetheless, the regulatory activities of CD1dhiCD5+ B cells from CD154TGCD22−/− mice were strikingly similar to those published for CD1dhiCD5+ B cells from WT mice [31], and B10 cells from CD154TGCD22−/− and WT mice produced similar levels of cytoplasmic IL-10 following ex vivo stimulation. Globally impaired IgG responses were also unique to CD154TGCD22−/− mice, arguing against constitutive CD40 signaling or continuous CD154 internalization, as also occurs in CD154TG mice (Figure 1A,F), as the explanation for this observation. It is also unlikely that B10 cells inhibited B cell differentiation directly since serum IgM levels were elevated in CD154TGCD22−/− mice. Thus, B10 cell depletion in WT mice and expansion in CD154TGCD22−/− mice had the predicted biologic effects of facilitating and inhibiting, respectively, IgG immune responses.

Agonistic CD40 mAb treatment in vivo “short-circuits” humoral immunity and impairs IgG production, GC formation, and B cell memory [13]. Tsubata and colleagues have also found accelerated termination of GC reactions to T cell-dependent Ags in hemizygous CD154TG mice, although IgG production remained robust [14], [15]. CD40 agonists also induce extrafollicular B cell differentiation. The “short circuiting” of IgG responses described above under robust CD40 ligation conditions (e.g., agonistic CD40 mAb) may be explained in part by the expansion and/or maturation of B10 and B10pro cells, as occurred in CD154TGCD22−/− mice. Consistent with this, B10 cells proliferate more rapidly than non-B10 cells following mitogen stimulation [27]. Therefore, the rapid expansion of Ag-specific B10 cells within extrafollicular foci of CD154TGCD22−/− mice may result in Ag consumption and/or clearance by IgM before the initiation of germinal center reactions and IgG production. Alternatively, B10 cells may indirectly regulate germinal center formation by altering TD immunity or the ability of dendritic cells to act as Ag-presenting cells during T cell activation [31], which are required for efficient B cell isotype switching.

Treatment of WT mice with the MB22-10 mAb that blocks CD22 ligand binding preferentially depleted spleen B10 and CD1dhiCD5+ B cells, but not follicular B cells (Figure 5A,B) as described [31]. While CD22 ligand binding is needed for the optimal survival of some B cell populations, such as MZ B cells [34], [35], [50], B10 cell development was normal in CD22−/− mice and dramatically expanded in CD154TGCD22−/− mice. As such, CD22 ligation with this particular mAb may induce signals that promote B10 cell apoptosis, alter their maturation, and/or affect their tissue distribution. B10 cell localization may be critical for function since spleen CD1dhiB220+ B cells were identified in both the MZ and follicular regions (Figure 1D), suggesting that B10 cells inhabit both sites. In support of this, spleen MZs were undetectable in CD22−/− mice, yet these mice had significant numbers of B10 and CD1dhiB220+ B cells scattered throughout their follicles. Intrafollicular CD1dhi B cells were especially prominent in CD154TGCD22−/− mice (Figure 1D). In any case, B cell depletion by the MB22-10 mAb is not dependent on Ab-dependent cellular cytotoxicity or complement activation [34]. In addition, the MB22-10 mAb impairs malignant B cell survival in mice [34]. Therefore, therapeutic B10 cell depletion may be useful for enhancing IgG responses, or for the treatment of tumors and immunosuppression.

Foxp3+CD25+ Treg cell numbers were normal in both CD154TG and CD154TGCD22−/− mice (Table 1). Similarly, B10 cells do not appear to directly regulate Treg cell numbers in an EAE model, where these two regulatory subsets function independently [31]. This suggests that the potential suppressive effect of B10 cells on IgG production did not result from Treg cell expansion. By contrast, the adoptive transfer of CD1dhiCD5+ B cells from wild type mice into CD19-deficient NZB/W F1 mice leads to a significant increase in Treg numbers [46]. While these results suggest that B10 cells could drive Treg cell expansion, the majority of spleen CD1dhiCD5+ B cells are not B10pro or B10 cells [27]. Thus, the adoptive transfer of non-B10 cells may independently promote CD4+ Treg cell generation. This possible explanation may also apply to studies suggesting that Breg cell-induced inhibition of inflammation is partly driven by Breg cell-induced Treg cell expansion. Nonetheless, spleen IL-10-competent T cell numbers were significantly increased in both CD154TG and CD154TGCD22−/− mice relative to WT and CD22−/− mice, in parallel with increased CD4+ and CD8+ memory T cell numbers (Table 1). The increase in IL-10-competent and memory T cells may therefore result from B cell CD154 expression. It is unlikely that the increase in IL-10-competent and memory T cells drives B10 cell expansion since B10 cell expansion was limited to CD154TGCD22−/− mice. Thus, B cell expression of CD154 appears to have intrinsic effects on B cells as well as extrinsic effects on cellular immunity.

T and B cell CD154 expression in SLE patients is proposed to drive autoimmunity [16], [17], [18], and a clear role for aberrant CD40 signaling in other inflammatory autoimmune diseases has emerged in recent years [51], [52]. It has also been suggested that chronic CD40 signaling, particularly in patients with SLE, functionally inactivates a subset of regulatory B cells [53]. However, the current studies suggest that B cell CD154 expression may predispose lupus patients towards enhanced B10 cell production. Consistent with this, B10pro cell numbers are expanded in SLE patients, and can be induced to mature and acquire IL-10 competence following agonistic CD40 stimulation [28]. Therefore, constitutive, robust CD40 signals may drive functional B10pro cell expansion and limit the pathogenic consequences of autoimmunity by reducing Ab isotype switching. Moreover, this study predicts that certain combinations of genetic traits that can individually induce autoimmune disease may actually reduce autoimmunity by expanding B10 cell numbers. For example, CD22−/− mice produce high-affinity serum IgG autoAbs spontaneously with age [54], and hemizygous CD154TG mice can develop relatively mild autoimmunity [19], [20]. B10 cell numbers are also expanded in type 1 diabetes-prone NOD mice, and in lupus-prone MRLlpr and NZB/W F1 mice, even prior to the appearance of disease [27], [38]. Similarly, blood B10pro cell numbers are expanded significantly in humans with lupus and other autoimmune diseases [28]. The observation that manipulation of the CD40 and CD22 signaling pathways can drive resident B10 cell expansion in vivo also highlights a potential therapeutic benefit for expanding B10 cells in humans to limit the pathogenic consequences of humoral IgG responses to self Ags.

Acknowledgments

We wish to thank Isaac G. Sanford for technical assistance in performing the experiments for these studies.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This work was supported by National Institutes of Health grants CA96547 and AI56363 (to TFT). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.